Increases in protein oxidation are associated with both normal aging and with age-associated neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Changes in the oxidative state of proteins, particularly at sulfhydryl groups, have been experimentally linked to both cell signaling events and to oxidative damage. Using a novel Redox 2D-PAGE proteomics technique to assay intermolecular disulfide bonds, as well as a new method with improved sensitivity to examine protein carbonylation, we will identify proteins whose oxidation state changes during both normal aging in the human and mouse central nervous system (CNS) and in AD brains from humans and transgenic mice as compared with age-matched controls. To determine which of these oxidatively modified proteins are involved in nerve cell death and which are involved in other signaling pathways, we will use a mouse hippocampal cell line wherein the cells can be killed by the generation of endogenous reactive oxygen species (ROS) or maintained in a viable state in the presence of prolonged, high levels of endogenous ROS. Importantly, we will also examine the effects of oxidative stress on the oxidation of extracellular (secreted) proteins in HT22 cells and in primary cultures of nerve and glia. The proteins that are oxidatively modified in these cultured cells will be compared to those that are modified during normal aging and in AD. These experiments will allow us to determine which subset of oxidatively modified proteins is involved in the cellular events that lead to nerve cell death. These proteins, in turn, will be used as targets for the identification of potentially therapeutic antioxidants that could be useful in preventing age-and disease-associated damage to the CNS.